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Background  Lactic acid bacteria (LAB) are one of the microorganisms of choice for the development of protein delivery systems for therapeutic purposes. Although there are numerous tools to facilitate genome engineering of lactobacilli; transformation efficiency still limits the ability to engineer their genomes. While genetically manipulating  Lactobacillus reuteri  ATCC PTA 6475 (LR 6475), we noticed that after an initial transformation, several LR 6475 strains significantly improved their ability to take up plasmid DNA via electroporation. Our goal was to understand the molecular basis for how these strains acquired the ability to increase transformation efficiency.    Results  Strains generated after transformation of plasmids pJP067 and pJP042 increased their ability to transform plasmid DNA about one million fold for pJP067, 100-fold for pSIP411 and tenfold for pNZ8048. Upon sequencing of the whole genome from these strains, we identified several genomic mutations and rearrangements, with all strains containing mutations in the transformation related gene A ( trgA ). To evaluate the role of  trgA  in transformation of DNA, we generated a  trg A null that improved the transformation efficiency of LR 6475 to transform pSIP411 and pJP067 by at least 100-fold, demonstrating that  trg A significantly impairs the ability of LR 6475 to take-up plasmid DNA. We also identified genomic rearrangements located in and around two prophages inserted in the LR 6475 genome that included deletions, insertions and an inversion of 336 Kb. A second group of rearrangements was observed in a Type I restriction modification system, in which the specificity subunits underwent several rearrangements in the target recognition domain. Despite the magnitude of these rearrangements in the prophage genomes and restriction modification systems, none of these genomic changes impacted transformation efficiency to the level induced by  trgA .    Conclusions  Our findings demonstrate how genetic manipulation of LR 6475 with plasmid DNA leads to genomic changes that improve their ability to transform plasmid DNA; highlighting  trgA  as the primary driver of this phenotype. Additionally, this study also underlines the importance of characterizing genetic changes that take place after genome engineering of strains for therapeutic purposes.    Electronic supplementary material  The online version of this article (10.1186/s12934-018-0986-8) contains supplementary material, which is available to authorized users.

Genome alterations associated with improved transformation efficiency in Lactobacillus reuteri